This collaborative project was initiated by Dr. Vesna A. Eterovic of the Universidad Central del Caribe, Puerto Rico. The purpose of this collaborative project is to use cembranoids to understand the functional architecture of the acetylcholine receptor (AChR). This understanding is basic to the field of protein structure, and also has medical applications for general anesthesia, and neuromuscular disorders. The nicotinic acetylcholine receptor (AChR) is the prototypical ligand-gated ion channel mediating synaptic transmission at the neuromuscular junction and between neurons in the peripheral and central nervous system. Neuronal receptors are altered in the brains of Alzheimer and Parkinson patients, while defects of peripheral receptors cause various myasthenias. This receptor protein is also the secondary site of action for local and general anesthetics. The inhibition of muscle AChR by anesthetics causes anesthesia related complications, particularly in patients suffering from Myasthenia Gravis where these complications are exacerbated by a reduced numbers of receptors. Cembranoids are a family of cyclic diterpenoids containing a 14-carbon cembrane ring substituted with hydroxy, epoxy or oxa-bridge groups, and often containing lactone rings. These cembranoids are effective non-competitive inhibitors of the AChR and over one hundred analogs have been isolated from Caribbean corals and characterized. High specific activity cembranoids are required to determine the location of the binding site and to characterize its reversible binding to the AChR. Competitive displacement of the binding by inhibitors, whose binding sites are known, would further specify the sites on the AChR. The AChR is a highly plastic protein where competitive inhibition between two ligands may result from steric as well as allosteric interactions. Specific questions to be addressed are: 1. Do cembranoids and phencyclidine (PCP) sites overlap or interact allosterically? 2. Do cembranoids bind to additional sites, not shared by PCP? 3. What is the relationship between cembranoid binding sites and those of pentobarbital, quinacrine, QX-222, ethidium bromide, histrionicotoxin, and tetracaine? Compounds in this group are known to bind to at least three topographically different sites, and preference for each receptor conformation is represented. In particular, pentobarbital and tetracaine bind to the resting state; quinacrine and QX-222 prefer the open channel conformation; ethidium bromide and PCP stabilize the desensitized state. Several cembranoids, such as the very active pseudoplexauric acid (PAME), contain an exocyclic double bond, activated by a nearby ester group. This double bond can be reacted with diazomethane to produce a pyrazoline derivative, which upon illumination would be converted to a cyclopropyl-PAME. Both the pyrazoline-PAME and cyclopropyl-PAME have been shown to be as active as PAME. Tritiated diazomethane at high specific activity has been recently developed at the NTLF. We have reacted PAME with tritiated diazomethane, and NMR analysis of that reaction indicated the presence of 3H-pyrazoline-PAME. The preliminary data indicates the conversion of the pyrazoline-PAME to cyclopropyl-PAME. Additional material will be required to achieve a complete characterization of these products.